There are many use environments, the fields of medical research and pharmaceutical development being examples, where it is necessary to accurately acquire fluid samples with volumes which may be as small as a few nanoliters. In these same fields, it is also often desirable to measure optical characteristics of the acquired fluid samples. Such optical characteristics include, for example, the ability of a sample to absorb light.
For instance, UV-Visible Spectrophotometry may be used to characterize the chemical composition of a liquid sample (in solution or suspension phase) using the absorbed spectra of the sample. The light absorbance of a sample depends on the pathlength L of light passing through the sample, as well as on the concentration of light absorbers (e.g., biomolecules, cells, etc) in a sample solution and the wavelength (λ) of light being used to characterize the sample. The wavelengths of UV-Visible light span from 200 nm to 800 nm, while ultraviolet wavelengths range from 200 to 400 nm.
UV-Visible spectrophotometry provides a way to determine the concentration, purity, and integrity of a biological sample without requiring additional sample preparation other than acquiring a sample. UV-Visible Spectrophotometry measurements depend on the light source (UV lamp), the sample and sampling technique. Most biological samples absorb electromagnetic radiation at wavelengths ranging from 200 nm to 800 nm, mostly 230, 260 and 280 nm. For a DNA or RNA sample in aqueous phase, one unit of absorbance 1 Å measured at a λ 260 nm and a pathlength of 10 mm is equal to 50/(40) ng/μl
Most biological samples are highly concentrated for down-stream process purpose (such as microarray spotting or protein sample preparation for mass spectrometer). The absorbance of such samples can be above the saturation limit for typical spectrophotometers if the pathlength is about 10 mm. While the sample concentration range can be extended by diluting the sample, diluting sample requires additional laboratory work and can result in errors.
Absorbance measurements on instruments, such as spectrophotometers, have a useful range where the measurement errors are minimal. As the absorbance approaches zero or the lower absorbance limit of the instrument (maximum transmittance), the uncertainties in the absorbance measurement are dominated by noise. It is difficult to measure a small change within a large signal. As the absorbance approaches infinity or the higher absorbance limit of the instrument (zero transmittance), the resulting signal has a lower amplitude than practical for measurement. In addition to the random errors inherent in measuring very low signals, stray light that reaches the detector of the instrument, limits the ability of a spectophotometer to measure at high absorbance, so that at a high concentration of sample, the absorbance measurement is lower than predicted.
Some conventional cuvetteless spectrophotometers can be used to measure the absorbance of small volume liquid samples (e.g., between 1-2 μl). These instruments typically provide a sample stage on which a sample droplet may be placed and an opposing surface which can be brought into contact with the droplet. By increasing the distance between the two surfaces, the droplet is stretched. Both the sample stage and the opposing surface are in communication with the respective ends of a source-side and detection-side optical fiber. Light from a light source passes through the source side optical fiber through the sample droplet to the detection-side fiber and is received by a detector within the instrument, permitting absorbance measurements of the sample.
However such instruments do not provide the capability for optimizing the absorbance measurement.